Methods and compositions for oral administration of proteins

ABSTRACT

This invention provides compositions comprising a protein, an absorption enhancer, a protease inhibitor, methods for treating diabetes mellitus, comprising administering same, and methods for oral administration of a protein with an enzymatic activity, comprising orally administering same.

FIELD OF INVENTION

This invention provides oral compositions comprising a protein, anabsorption enhancer, and a protease inhibitor and a method foradministering same.

BACKGROUND OF THE INVENTION

Due to improved biotechnology, the accessibility of biologically activepeptides to the pharmaceutical industry has increased considerably.However, a limiting factor in the development of peptide drugs is therelative ineffectiveness when given perorally. Almost all peptide drugsare parenterally administered, although parenterally administeredpeptide drugs are often connected with low patient compliance.

Insulin is a medicament used to treat patients suffering from diabetes,and is the only treatment for insulin-dependent diabetes mellitus.Diabetes Mellitus is characterized by a pathological condition ofabsolute or relative insulin deficiency, leading to hyperglycemia, andis one of the main threats to human health in the 21st century. Theglobal figure of people with diabetes is set to rise to 220 million in2010, and 300 million in 2025. Type I diabetes is caused primarily bythe failure of the pancreas to produce insulin. Type II diabetes,involves a lack of responsiveness of the body to the action of insulin.

Approximately 20%-30% of all diabetics use daily insulin injections tomaintain their glucose levels. An estimated 10% of all diabetics aretotally dependent on insulin injections.

Currently, the only route of insulin administration is injection. Dailyinjection of insulin is causes considerable suffering for patients. Sideeffects such as lipodystrophy at the site of the injection, lipatrophy,lilpohypertrophy, and occasional hypoglycemia are known to occur. Inaddition, subcutaneous administration of insulin does not typicallyprovide the fine continuous regulation of metabolism that occursnormally with insulin secreted from the pancreas directly into the livervia the portal vein.

The present invention addresses the need for an alternate solution foradministration of insulin and peptides such as insulin.

SUMMARY OF THE INVENTION

This invention provides, in one embodiment, a composition comprising aprotein having a molecular weight of up to 100,000 Daltons, a proteaseinhibitor, and an absorption enhancer, wherein the absorption enhancerenhances the absorption of a protein through an intestinal mucosalbarrier.

In another embodiment, the present invention provides a compositioncomprising a protein having a molecular weight of up to 100,000 Daltons,a protease inhibitor, and SNAC or SNAD.

In another embodiment, the present invention provides a method for oraladministration of a protein having a molecular weight up to 100,000Daltons to a subject, whereby a substantial fraction of said proteinretains its activity after absorption, through an intestinal mucosalbarrier of a subject, comprising administering orally to a subject apharmaceutical composition comprising a protein, a protease inhibitor,and an absorption enhancer, wherein an absorption enhancer enhances theabsorption of a protein through an intestinal mucosal barrier.

In another embodiment, the present invention provides a method fortreating diabetes mellitus in a subject, comprising administering orallyto a subject a pharmaceutical composition comprising insulin, Exenatide,or a combination thereof a protease inhibitor, and a absorptionenhancer, wherein an absorption enhancer enhances the absorption of aprotein through an intestinal mucosal barrier, thereby treating diabetesmellitus.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the changes in blood glucose levels aftertreatment with the formulations of the invention.

FIG. 2 is a graph showing the changes in blood C-peptide levels aftertreatment with the formulations of the invention.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides compositions and methods comprising a protein, aprotease inhibitor, and a absorption enhancer. In another embodiment,the present invention provides compositions and methods comprising aprotein having a molecular weight of up to 100,000 Daltons, a proteaseinhibitor, and a absorption enhancer. In another, embodiment, theabsorption enhancer enhances the absorption of the protein through anintestinal mucosal barrier. In another, embodiment, the absorptionenhancer enhances the absorption of a protein having a molecular weightof up to 100,000 Daltons through an intestinal mucosal barrier.

In another embodiment, the compositions of the present invention includean active agent, a protease inhibitor, and a carrier. In anotherembodiment, the compositions of the present invention are used todeliver various proteins through various biological, chemical, andphysical barriers and are particularly suited for delivering proteinswhich are subject to environmental degradation. In another embodiment,the compositions of the present invention are used to deliver activeproteins through various biological, chemical, and physical barriers andare particularly suited for delivering active proteins which are subjectto environmental degradation. In another embodiment, the compositionscomprise an active protein, SNAC, a protease inhibitor, and EDTA orNa-EDTA. In another embodiment, the compositions comprise an activeprotein, SNAC, a protease inhibitor, and an omega-3 fatty acid. Inanother embodiment, the compositions comprise an active protein, SNAC, aprotease inhibitor, EDTA or Na-EDTA, and an omega-3 fatty acid. Inanother embodiment, the composition as described herein is an oralpharmaceutical composition. In another embodiment, the composition asdescribed herein is an encapsulated oral pharmaceutical composition. Inanother embodiment, the composition as described herein is in a liquidoral dosage form. In another embodiment, the composition as describedherein is in a dry oral dosage form (tablet etc.).

In another, embodiment, the protein of the present invention has amolecular weight of 1,000-5,000 Daltons. In another, embodiment, theprotein of the present invention has a molecular weight of 5,000-10,000Daltons. In another, embodiment, the protein of the present inventionhas a molecular weight of 10,000-20,000 Daltons. In another, embodiment,the protein of the present invention has a molecular weight of20,000-30,000 Daltons. In another, embodiment, the protein of thepresent invention has a molecular weight of 40,000-50,000 Daltons. Inanother, embodiment, the protein of the present invention has amolecular weight of 50,000-60,000 Daltons. In another, embodiment, theprotein of the present invention has a molecular weight of 60,000-70,000Daltons. In another, embodiment, the protein of the present inventionhas a molecular weight of 70,000-80,000 Daltons. In another, embodiment,the protein of the present invention has a molecular weight of80,000-90,000 Daltons. In another, embodiment, the protein of thepresent invention has a molecular weight of 90,000-100,000 Daltons. Inanother, embodiment, the protein of the present invention has amolecular weight of 100,000-150,000 Daltons.

In another embodiment, the protein has a molecular weight (MW) of 1-50kilodalton (kDa). In another embodiment, the MW is 1-45 kDa. In anotherembodiment, the MW is 1-40 kDa. In another embodiment, the MW is 1-35kDa. In another embodiment, the MW is 1-30 kDa. In another embodiment,the MW is 1-25 kDa. In another embodiment, the MW is 1-20 kDa. Inanother embodiment, the MW is 10-50 kDa. In another embodiment, the MWis 15-50 kDa. In another embodiment, the MW is 20-50 kDa. In anotherembodiment, the MW is 25-50 kDa. In another embodiment, the MW is 30-50kDa. In another embodiment, the MW is 35-50 kDa. In another embodiment,the MW is 1-100 kDa. In another embodiment, the MW is 1-90 kDa. Inanother embodiment, the MW is 1-80 kDa. In another embodiment, the MW is1-70 kDa. In another embodiment, the MW is 1-60 kDa. In anotherembodiment, the MW is 10-100 kDa. In another embodiment, the MW is15-100 kDa. In another embodiment, the MW is 20-100 kDa. In anotherembodiment, the MW is 25-100 kDa. In another embodiment, the MW is30-100 kDa. In another embodiment, the MW is 10-80 kDa. In anotherembodiment, the MW is 15-80 kDa. In another embodiment, the MW is 20-80kDa. In another embodiment, the MW is 25-80 kDa. In another embodiment,the MW is 30-80 kDa. Each possibility represents a separate embodimentof the present invention.

In another embodiment, the MW is less than 20 kDa. In anotherembodiment, the MW is less than 25 kDa. In another embodiment, the MW isless than 30 kDa. In another embodiment, the MW is less than 35 kDa. Inanother embodiment, the MW is less than 40 kDa. In another embodiment,the MW is less than 45 kDa. In another embodiment, the MW is less than50 kDa. In another embodiment, the MW is less than 55 kDa. In anotherembodiment, the MW is less than 60 kDa. In another embodiment, the MW isless than 65 kDa. In another embodiment, the MW is less than 70 kDa. Inanother embodiment, the MW is less than 75 kDa. In another embodiment,the MW is less than 80 kDa. In another embodiment, the MW is less than85 kDa. In another embodiment, the MW is less than 90 kDa. In anotherembodiment, the MW is less than 95 kDa. In another embodiment, the MW isless than 100 kDa.

In another, embodiment, the protein of the present invention is insulin.In another embodiment, the insulin of methods and compositions of thepresent invention is human insulin. In another embodiment, the insulinis recombinant insulin. In another embodiment, the insulin isrecombinant human insulin. In another embodiment, the insulin is bovineinsulin. In another embodiment, the insulin is porcine insulin. Inanother embodiment, the insulin is whale insulin. In another embodiment,the insulin is a metal complex of insulin (e.g. a zinc complex ofinsulin, protamine zinc insulin, or globin zinc).

In another embodiment, the insulin is endogenous insulin. In anotherembodiment, the insulin is fast-acting insulin. In another embodiment,the insulin is lente insulin. In another embodiment, the insulin issemilente insulin. In another embodiment, the insulin is ultralenteinsulin. In another embodiment, the insulin is NPH insulin. In anotherembodiment, the insulin is glargine insulin. In another embodiment, theinsulin is lispro insulin. In another embodiment, the insulin is aspartinsulin. In another embodiment, the insulin is a combination of two ormore of any of the above types of insulin. In another embodiment, theinsulin is any other type of insulin known in the art. Each possibilityrepresents a separate embodiment of the present invention.

In one embodiment, the amount of insulin utilized in the methods andcompositions of the present invention is 0.5-3 units (u)/kg in humans.In one embodiment, the units used to measure insulin in methods andcompositions of the present invention are USP Insulin Units. In oneembodiment, the units used to measure insulin are milligrams. In anotherembodiment, one international Unit (IU) of Insulin is equivalent to 45.5mg insulin.

In another embodiment, the amount of insulin is 0.1-1 u/kg. In anotherembodiment, the amount is 0.2-1 u/kg. In another embodiment, the amountis 0.3-1 u/kg. In another embodiment, the amount is 0.5-1 u/kg. Inanother embodiment, the amount is 0.1-2 u/kg. In another embodiment, theamount is 0.2-2 u/kg. In another embodiment, the amount is 0.3-2 u/kg.In another embodiment, the amount is 0.5-2 u/kg. In another embodiment,the amount is 0.7-2 u/kg. In another embodiment, the amount is 1-2 u/kg.

In another embodiment, the amount is 1.2-2 u/kg. In another embodiment,the amount is 1-1.2 u/kg. In another embodiment, the amount is 1-1.5u/kg. In another embodiment, the amount is 1-2.5 u/kg. In anotherembodiment, the amount is 1-3 u/kg. In another embodiment, the amount is2-3 u/kg. In another embodiment, the amount is 1-5 u/kg. In anotherembodiment, the amount is 2-5 u/kg. In another embodiment, the amount is3-5 u/kg.

In another embodiment, the amount of insulin is 0.1 u/kg. In anotherembodiment, the amount is 0.2 u/kg. In another embodiment, the amount is0.3 u/kg. In another embodiment, the amount is 0.4 u/kg. In anotherembodiment, the amount is 0.5 u/kg. In another embodiment, the amount is0.6 u/kg. In another embodiment, the amount is 0.8 u/kg. In anotherembodiment, the amount is 1 u/kg. In another embodiment, the amount is1.2 u/kg. In another embodiment, the amount is 1.4 u/kg. In anotherembodiment, the amount is 1.6 u/kg. In another embodiment, the amount is1.8 u/kg. In another embodiment, the amount is 2 u/kg. In anotherembodiment, the amount is 2.2 u/kg. In another embodiment, the amount is2.5 u/kg. In another embodiment, the amount is 3 u/kg.

In another embodiment, the amount of insulin is 1-10 u. In anotherembodiment, the amount is 2-10 u. In another embodiment, the amount is3-10 u. In another embodiment, the amount is 5-10 u. In anotherembodiment, the amount is 1-20 u. In another embodiment, the amount is2-20 u. In another embodiment, the amount is 3-20 u. In anotherembodiment, the amount is 5-20 u. In another embodiment, the amount is7-20 u. In another embodiment, the amount is 10-20 u. In anotherembodiment, the amount is 12-20 u. In another embodiment, the amount is10-12 u. In another embodiment, the amount is 10-15 u. In anotherembodiment, the amount is 10-25 u. In another embodiment, the amount is10-30 u. In another embodiment, the amount is 20-30 u. In anotherembodiment, the amount is 10-50 u. In another embodiment, the amount is20-50 u. In another embodiment, the amount is 30-50 u. In anotherembodiment, the amount is 20-100 u. In another embodiment, the amount is30-100 u. In another embodiment, the amount is 100-150 u. In anotherembodiment, the amount is 100-250 u. In another embodiment, the amountis 100-300 u. In another embodiment, the amount is 200-300 u. In anotherembodiment, the amount is 100-500 u. In another embodiment, the amountis 200-500 u. In another embodiment, the amount is 300-500 u. In anotherembodiment, the amount is 200-1000 u. In another embodiment, the amountis 300-1000 u.

In another embodiment, the amount of insulin is 1 u. In anotherembodiment, the amount is 2 u. In another embodiment, the amount is 3 u.In another embodiment, the amount is 4 u. In another embodiment, theamount is 5 u. In another embodiment, the amount is 6 u. In anotherembodiment, the amount is 8 u. In another embodiment, the amount is 10u. In another embodiment, the amount is 12 u. In another embodiment, theamount is 14 u. In another embodiment, the amount is 16 u. In anotherembodiment, the amount is 18 u. In another embodiment, the amount is 20u. In another embodiment, the amount is 22 u. In another embodiment, theamount is 25 u. In another embodiment, the amount is 30 u. In anotherembodiment, the amount is 50 u. In another embodiment, the amount is 80u. In another embodiment, the amount is 100 u. In another embodiment,the amount is 120 u. In another embodiment, the amount is 140 u. Inanother embodiment, the amount is 160 u. In another embodiment, theamount is 180 u. In another embodiment, the amount is 200 u. In anotherembodiment, the amount is 300 u. In another embodiment, the amount is500 u.

In another embodiment, the protein is an insulin secretagogue. Inanother embodiment, the protein is GLP-1. In another embodiment, theprotein is a GLP-1 analogue. In another embodiment, the protein is aGLP-1 mimetic. In another embodiment, the protein is an incretinmimetic. In another embodiment, the protein mimics the GLP-1 incretin.In another embodiment, the protein is GLP-2. In another embodiment, theprotein is a GLP-2 analogue. In another embodiment, the protein is aGLP-2 mimetic.

In another embodiment, the protein is Exenatide. In another embodiment,the oral formulations of the present invention protect Exenatidebreakdown in the stomach. In another embodiment, Exenatide formulationof the invention controls blood sugar levels. In another embodiment,Exenatide formulation of the invention helps control blood sugar levels.In another embodiment, Exenatide formulation of the invention inducespancreatic production of insulin. In another embodiment, Exenatideformulation of the invention is used to treat type-2 (non-insulindependent) diabetes. In another embodiment, Exenatide formulation of theinvention is used in conjunction with other diabetes medicines.

In another embodiment, the amount of Exenatide in a formulation asdescribed herein is 10 mcg to 1 mg. In another embodiment, the amount ofExenatide in a formulation as described herein is 10 mcg to 25 mcg. Inanother embodiment, the amount of Exenatide in a formulation asdescribed herein is 25 mcg to 50 mcg. In another embodiment, the amountof Exenatide in a formulation as described herein is 50 mcg to 60 mcg.In another embodiment, the amount of Exenatide in a formulation asdescribed herein is 60 mcg to 70 mcg. In another embodiment, the amountof Exenatide in a formulation as described herein is 70 mcg to 80 mcg.In another embodiment, the amount of Exenatide in a formulation asdescribed herein is 80 mcg to 90 mcg. In another embodiment, the amountof Exenatide in a formulation as described herein is 90 mcg to 100 mcg.In another embodiment, the amount of Exenatide in a formulation asdescribed herein is 100 mcg to 110 mcg. In another embodiment, theamount of Exenatide in a formulation as described herein is 110 mcg to125 mcg. In another embodiment, the amount of Exenatide in a formulationas described herein is 125 mcg to 150 mcg. In another embodiment, theamount of Exenatide in a formulation as described herein is 150 mcg to175 meg. In another embodiment, the amount of Exenatide in a formulationas described herein is 175 mcg to 200 mcg. In another embodiment, theamount of Exenatide in a formulation as described herein is 200 mcgto220 mcg. In another embodiment, the amount of Exenatide in aformulation as described herein is 220 mcg to 240 mcg. In anotherembodiment, the amount of Exenatide in a formulation as described hereinis 240 mcg to 260 mcg. In another embodiment, the amount of Exenatide ina formulation as described herein is 260 mcg to 300 mcg.

In another embodiment, the amount of Exenatide in a formulation asdescribed herein is 300 mcg to 350 mcg. In another embodiment, theamount of Exenatide in a formulation as described herein is 350 mcg to400 mcg. In another embodiment, the amount of Exenatide in a formulationas described herein is 400 mcg to 450 mcg. In another embodiment, theamount of Exenatide in a formulation as described herein is 450 mcg to500 mcg. In another embodiment, the amount of Exenatide in a formulationas described herein is 550 mcg to 600 mcg. In another embodiment, theamount of Exenatide in a formulation as described herein is 600 mcg to700 mcg. In another embodiment, the amount of Exenatide in a formulationas described herein is 700 mcg to 800 mcg. In another embodiment, theamount of Exenatide in a formulation as described herein is 800 mcg to900 mcg. In another embodiment, the amount of Exenatide in a formulationas described herein is 900 mcg to 1 mg.

In another embodiment, the Exenatide formulation as described herein istaken twice a week. In another embodiment, the Exenatide formulation asdescribed herein is taken once every two days. In another embodiment,the Exenatide formulation as described herein is taken once a day. Inanother embodiment, the Exenatide formulation as described herein istaken twice a day. In another embodiment, the Exenatide formulation asdescribed herein is taken three times a day. In another embodiment, theExenatide formulation as described herein is taken four times a day. Inanother embodiment, the Exenatide formulation as described herein istaken five times a day. In another embodiment, one of skill in the artdetermines the dosage of a Exenatide formulation as described herein. Inanother embodiment, one of skill in the art determines the daily dose ofa Exenatide formulation as described herein. In another embodiment, oneof skill in the art determines the daily dosing regimen of a Exenatideformulation as described herein.

In another embodiment, the Exenatide formulation as described herein istaken at least 15 minutes before a meal. In another embodiment, theExenatide formulation as described herein is taken at least 30 minutesbefore a meal. In another embodiment, the Exenatide formulation asdescribed herein is taken at least 45 minutes before a meal. In anotherembodiment, the Exenatide formulation as described herein is taken atleast 60 minutes before a meal. In another embodiment, the Exenatideformulation as described herein is taken at least 75 minutes before ameal. In another embodiment, the Exenatide formulation as describedherein is taken at least 90 minutes before a meal. In anotherembodiment, the Exenatide formulation as described herein is taken atleast 100 minutes before a meal. In another embodiment, the Exenatideformulation as described herein is taken at least 120 minutes before ameal. In another embodiment, the Exenatide formulation as describedherein is taken at least 150 minutes before a meal. In anotherembodiment, the Exenatide formulation as described herein is taken atleast 180 minutes before a meal.

In another embodiment, the Exenatide formulation as described hereinreduces the side effects associated with an injectable dosage formcomprising Exenatide. In another embodiment, the Exenatide formulationas described herein reduces nausea as a side effect which is associatedwith an injectable dosage form comprising Exenatide. In anotherembodiment, the Exenatide formulation as described herein does notinduce nausea as a side effect which is associated with an injectabledosage form comprising Exenatide.

In another embodiment, the protein is glucagon. In another embodiment,the protein is interferon gamma. In another embodiment, the protein isinterferon alpha. In another embodiment, the protein is a growthhormone. In another embodiment, the protein is erythropoietin. Inanother embodiment, the protein is granulocyte colony stimulating factor(G-CSF). In another embodiment, the protein is omentin. In anotherembodiment, the protein is calcitonin. In another embodiment, theprotein is PTH. In another embodiment, the protein is PYY.

In another embodiment, the protein is any other protein known in theart. In another embodiment, the protein is a growth hormone. In oneembodiment, the growth hormone is somatotropin. In another embodiment,the growth hormone is Insulin Growth Factor-I (IGF-I). In anotherembodiment, the growth hormone is any other growth hormone known in theart.

In another embodiment, methods and compositions of the present inventionare used to administer a human serum albumin. Human serum albumin isnot, in one embodiment, considered to be a pharmaceutically-activecomponent; however, it can be used in the context of the presentinvention as a therapeutically-beneficial carrier for an activecomponent. Each type of protein represents a separate embodiment of thepresent invention.

In one embodiment, the protein is an enzyme. In some embodiments, theprotein is a receptor ligand, transporter, or a storage protein. In oneembodiment, the protein is a structural protein.

In some embodiments, the enzyme is an oxidoreductase, transferase,hydrolase, lyase, isomerase, or ligase. In some embodiments,oxidoreductases act on the aldehyde or oxo group of donors, on the CH—CHgroup of donors, on the CH—NH(2) group of donors, on the CH—NH group ofdonors, on NADH or NADPH, on the CH—OH group of donors, on nitrogenouscompounds as donors, on a sulfur group of donors, on a heme group ofdonors, on diphenols and related substances as donors, on a peroxide asacceptor, on hydrogen as donor, on single donors with incorporation ofmolecular oxygen, on paired donors, on superoxide as acceptor, oxidizingmetal ions, on CH or CH(2) groups, on iron-sulfur proteins as donors, onreduced flavodoxin as donor, on phosphorus or arsenic in donors, or onx-H and y-H to form an x-y bond.

In some embodiments, transferases are acyltransferases orglycosyltransferases. In some embodiments, transferases transferaldehyde or ketone residues. In some embodiments, transferases transferalkyl or aryl groups, other than methyl groups. In some embodiments,transferases transfer nitrogenous, phosphorous, sulfur or seleniumcontaining groups.

In some embodiments, hydrolases are glycosylases or act on ether bonds,on peptide bonds, on carbon-nitrogen bonds, other than peptide bonds, onacid anhydrides, on carbon-carbon bonds, on halide bonds, onphosphorus-nitrogen bonds, on sulfur-nitrogen bonds, oncarbon-phosphorus bonds, on sulfur-sulfur bonds, or on carbon-sulfurbonds.

In some embodiments, lyases are carbon-carbon lyases, carbon-oxygenlyases, carbon-nitrogen lyases, carbon-sulfur lyases, carbon-halidelyases, phosphorus-oxygen lyases, or other lyases.

In some embodiments, isomerases are racemases or epimerases,cis-trans-isomerases, intramolecular oxidoreductases, intramoleculartransferases, intramolecular lyases, or other isomerases.

In some embodiments, ligases form carbon-sulfur bonds, carbon-nitrogenbonds, carbon-carbon bonds, phosphoric ester bonds, or nitrogen-metalbonds.

In some embodiments, transporter proteins are annexins, ATP-bindingcassette transporters, hemoglobin, ATPases, calcium channels, potassiumchannels, sodium channels, or solute carriers.

In some embodiments, storage proteins comprise albumins, lactoglobulins,casein ovomucin, ferritin, phosvitin, lactoferrin, or vitellogenin. Inone embodiment, albumins comprise avidin, ovalbumin, serum albumin,parvalbumin, c-reactive proteinprealbumin, conalbumin, ricin,lactalbumin, methemalbumin, or transthyretin.

In some embodiments, structural proteins comprise amyloid, collagenelastin, or fibrillin.

In some embodiments, the protein is a viral protein, bacterial protein,invertebrate protein, or vertebrate protein. In some embodiments, theprotein is a recombinant protein. In one embodiment, the protein is arecombinant protein. In one embodiment, the recombinant protein is arecombinant human protein.

The molecular weights of some of the proteins mentioned above are asfollows: insulin—6 kilodalton (kDa); glucagon—3.5 kDa; interferon, 28kDa, growth hormone—21.5-47 kDa; human serum albumin—69 kDa;erythropoietin—34 kDa; G-CSF—30-34 kDa. Thus, in one embodiment, themolecular weight of these proteins is appropriate for administration bymethods of the present invention.

As provided herein, a protease inhibitor protects the protein of thepresent invention from cleavage. In another embodiment, the presentinvention provides that a protease inhibitor protects insulin of thepresent invention from cleavage. In another, embodiment, the presentinvention provides that a protease inhibitor facilitates the proteinabsorption in the intestine of a subject. In another, embodiment, thepresent invention provides that a protease inhibitor facilitates theabsorption of insulin in the intestine of a subject.

In another, embodiment, a serpin is: Alpha 1-antitrypsin,Antitrypsin-related protein, Alpha 1-antichymotrypsin, Kallistatin,Protein C inhibitor, Cortisol binding globulin, Thyroxine-bindingglobulin, Angiotensinogen, Centerin, Protein Z-related proteaseinhibitor, Vaspin, Monocyte neutrophil elastase inhibitor, Plasminogenactivator inhibitor-2, Squamous cell carcinoma antigen-1 (SCCA-1),Squamous cell carcinoma antigen-2 (SCCA-2), Maspin, PI-6, Megsin, PI-8,PI-9, Bomapin, Yukopin, Hurpin/Headpin, Antithrombin, Heparin cofactorII, Plasminogen activator inhibitor 1, Glia derived nexin/Protease nexinI, Pigment epithelium derived factor, Alpha 2-antiplasmin, Complement1-inhibitor, 47 kDa Heat shock protein (HSP47), Neuroserpin, or Pancpin.

In another embodiment, the present invention provides that the proteaseinhibitor is a trypsin inhibitor such as but not limited to: Lima beantrypsin inhibitor, Aprotinin, soy bean trypsin inhibitor (SBTI), orOvomucoid.

In another embodiment, the present invention provides that the proteaseinhibitor is a Cysteine protease inhibitor. In another, embodiment, thepresent invention provides that Cysteine protease inhibitors of theinvention comprise: cystatin, type 1 cystatins (or stefins), Cystatinsof type 2,human cystatins C, D, S, SN, and SA, cystatin E/M, cystatin F,type 3 cystatins, or kininogens.

In another embodiment, the present invention provides that the proteaseinhibitor is a Threonine protease inhibitor. In another, embodiment, thepresent invention provides that Threonine protease inhibitors of theinvention comprise: Bortezomib, MLN-519, ER-807446, TMC-95A.

In another embodiment, the present invention provides that the proteaseinhibitor is an Aspartic protease inhibitor. In another, embodiment, thepresent invention provides that Aspartic protease inhibitors of theinvention comprise: α₂-Macroglobulin, Pepstatin A, Aspartic proteaseinhibitor 11, Aspartic protease inhibitor 1, Aspartic protease inhibitor2, Aspartic protease inhibitor 3, Aspartic 2 0 protease inhibitor 4,Aspartic protease inhibitor 5, Aspartic protease inhibitor 6, Asparticprotease inhibitor 7, Aspartic protease inhibitor 8, Aspartic proteaseinhibitor 9, Pepsin inhibitor Dit33, Aspartyl protease inhibitor, orProtease A inhibitor 3.

In another embodiment, the present invention provides that the proteaseinhibitor is a Metalloprotease inhibitor. In another, embodiment, thepresent invention provides that Metalloprotease inhibitors of theinvention comprise: Angiotensin-1 -converting enzyme inhibitory peptide,Antihemorragic factor BJ46a, Beta-casein, Proteinase inhibitor CeKI,Venom metalloproteinase inhibitor DM43, Carboxypeptidase A inhibitor,smpI, IMPI, Alkaline proteinase, inh, Latexin, Carboxypeptidaseinhibitor, Antihemorragic factor HSF, Testican-3, SPOCK3, TIMP1,Metalloproteinase inhibitor 1, Metalloproteinase inhibitor 2, TIMP2,Metalloproteinase inhibitor 3, TIMP3, Metalloproteinase inhibitor 4,TIMP4, Putative metalloproteinase inhibitor tag-225, Tissue inhibitor ofmetalloprotease, WAP, kazal, immunoglobulin, or kunitz and NTRdomain-containing protein 1.

In some embodiments, a protease inhibitor is a suicide inhibitor, atransition state inhibitor, or a chelating agent. In some embodiments,the protease inhibitor of the present invention is: AEBSF-HC1,(epsilon)-aminocaproic acid,(alpha) 1-antichymotypsin, antipain,antithrombin III,(alpha) 1-antitrypsin ([alpha] 1-proteinaseinhibitor),APMSF-HC1 (4-amidinophenyl-methane sulfonyl-fluoride),sprotinin, benzamidine-HC1,chymostatin, DFP(diisopropylfluoro-phosphate), leupeptin, PEFABLOC® SC(4-(2-Aminoethyl)-benzenesulfonyl fluoride hydrochloride), PMSF(phenylmethyl sulfonyl fluoride), TLCK(1-Chloro-3-tosylamido-7-amino-2-heptanone HC1), TPCK (1-Chloro-3- tosylamido-4-phenyl-2-butanone), pentamidine isethionate, pepstatin,guanidium, alpha2-macroglobulin, a chelating agent of zinc, oriodoacetate, zinc. Each possibility represents a separate embodiment ofthe present invention.

In another embodiment, the amount of a protease inhibitor utilized inmethods and compositions of the present invention is 0.1 mg/dosage unit.In another embodiment, the amount of a protease inhibitor is 0.2mg/dosage unit. In another embodiment, the amount is 0.3 mg/dosage unit.In another embodiment, the amount is 0.4 mg/dosage unit. In anotherembodiment, the amount is 0.6 mg/dosage unit. In another embodiment, theamount is 0.8 mg/dosage unit. In another embodiment, the amount is 1mg/dosage unit. In another embodiment, the amount is 1.5 mg/dosage unit.In another embodiment, the amount is 2 mg/dosage unit. In anotherembodiment, the amount is 2.5 mg/dosage unit. In another embodiment, theamount is 3 mg/dosage unit. In another embodiment, the amount is 5mg/dosage unit. In another embodiment, the amount is 7 mg/dosage unit.In another embodiment, the amount is 10 mg/dosage unit. In anotherembodiment, the amount is 12 mg/dosage unit. In another embodiment, theamount is 15 mg/dosage unit. In another embodiment, the amount is 20mg/dosage unit. In another embodiment, the amount is 30 mg/dosage unit.In another embodiment, the amount is 50 mg/dosage unit. In anotherembodiment, the amount is 70 mg/dosage unit. In another embodiment, theamount is 100 mg/dosage unit.

In another embodiment, the amount of a protease inhibitor is 0.1-1mg/dosage unit. In another embodiment, the amount of a proteaseinhibitor is 0.2-1 mg/dosage unit. In another embodiment, the amount is0.3-1 mg/dosage unit. In another embodiment, the amount is 0.5-1mg/dosage unit. In another embodiment, the amount is 0.1-2 mg/dosageunit. In another embodiment, the amount is 0.2-2 mg/dosage unit. Inanother embodiment, the amount is 0.3-2 mg/dosage unit. In anotherembodiment, the amount is 0.5-2 mg/dosage unit. In another embodiment,the amount is 1-2 mg/dosage unit. In another embodiment, the amount is1-10 mg/dosage unit. In another embodiment, the amount is 2-10 mg/dosageunit. In another embodiment, the amount is 3-10 mg/dosage unit. Inanother embodiment, the amount is 5-10 mg/dosage unit. In anotherembodiment, the amount is 1-20 mg/dosage unit. In another embodiment,the amount is 2-20 mg/dosage unit. In another embodiment, the amount is3-20 mg/dosage unit. In another embodiment, the amount is 5-20 mg/dosageunit. In another embodiment, the amount is 10-20 mg/dosage unit. Inanother embodiment, the amount is 10-100 mg/dosage unit. In anotherembodiment, the amount is 20-100 mg/dosage unit. In another embodiment,the amount is 30-100 mg/dosage unit. In another embodiment, the amountis 50-100 mg/dosage unit. In another embodiment, the amount is 10-200mg/dosage unit. In another embodiment, the amount is 20-200 mg/dosageunit. In another embodiment, the amount is 30-200 mg/dosage unit. Inanother embodiment, the amount is 50-200 mg/dosage unit. In anotherembodiment, the amount is 100-200 mg/dosage unit.

In another embodiment, the amount of a protease inhibitor utilized inthe methods and compositions of the present invention is 1000 k.i.u.(kallikrein inactivator units)/ dosage unit. In another embodiment, theamount is 10 k.i.u./dosage unit. In another embodiment, the amount is 12k.i.u./dosage unit. In another embodiment, the amount is 15k.i.u./dosage unit. In another embodiment, the amount is 20k.i.u./dosage unit. In another embodiment, the amount is 30k.i.u./dosage unit. In another embodiment, the amount is 40k.i.u./dosage unit. In another embodiment, the amount is 50k.i.u./dosage unit. In another embodiment, the amount is 70k.i.u./dosage unit. In another embodiment, the amount is 100k.i.u./dosage unit. In another embodiment, the amount is 150k.i.u./dosage unit. In another embodiment, the amount is 200k.i.u./dosage unit. In another embodiment, the amount is 300k.i.u./dosage unit. In another embodiment, the amount is 500k.i.u./dosage unit. In another embodiment, the amount is 700k.i.u./dosage unit. In another embodiment, the amount is 1500k.i.u./dosage unit. In another embodiment, the amount is 3000k.i.u./dosage unit. In another embodiment, the amount is 4000k.i.u./dosage unit. In another embodiment, the amount is 5000k.i.u./dosage unit. Each amount of a first or a second proteaseinhibitor represents a separate embodiment of the present invention.

In another embodiment, the amount is 50-5000 k.i.u./dosage unit. Inanother embodiment, the amount is 500-4000 k.i.u./dosage unit. Inanother embodiment, the amount is 100-1000 k.i.u./dosage unit. Inanother embodiment, the amount is 200-1500 k.i.u./dosage unit.

In another embodiment, compositions of the present invention comprise asubstance that enhances absorption of a protein of the invention throughan intestinal mucosal barrier. In another embodiment, compositions ofthe present invention further comprise a substance that enhancesabsorption of insulin through an intestinal mucosal barrier. In anotherembodiment, compositions of the present invention further comprise asubstance that enhances absorption of Exenatide through an intestinalmucosal barrier. In another embodiment, compositions of the presentinvention further comprise a substance that reduces the degradation ofExenatide in the digestive system.In another embodiment, compositions ofthe present invention further comprise a substance that reduces thedegradation of Exenatide in the stomach. In another embodiment,compositions of the present invention further comprise a substance thatreduces the degradation of Exenatide in the intestine. Such a substanceis referred to herein as an “enhancer.” As provided herein, enhancers,when used together with omega-3 fatty acids or a protease inhibitor,enhance the ability of a proitein to be absorbed in the intestine. Asprovided herein, enhancers, when used together with omega-3 fatty acidsand a protease inhibitor, enhance the ability of insulin to be absorbedin the intestine. As provided herein, enhancers, when used together withomega-3 fatty acids and a protease inhibitor, enhance the ability ofExenatide to be absorbed in the intestine.

In one embodiment, the enhancer is didecanoylphosphatidylcholine (DDPC).In one embodiment, the enhancer is a chelating agent such asethylenediaminetetraacetic acid (EDTA) or egtazic acid EGTA. In anotherembodiment, EDTA is sodium-EDTA. In some embodiments, the enhancer is NOdonor. In some embodiments, the enhancer is a bile acid,glycine-conjugated form of a bile acid, or an alkali metal salt. In oneembodiment, absorption enhancement is achieved through utilization of acombination of a-galactosidase and β-mannanase.In some embodiments, theenhancer is a fatty acid such as sodium caprate. In one embodiment, theenhancer is sodium glycocholate. In one embodiment, the enhancer issodium salicylate. In one embodiment, the enhancer isn-dodecyl-β-D-maltopyranoside. In some embodiments, surfactants serve asabsorption enhancer. In one embodiment, the enhancer is chitisan such asN,N,N-trimethyl chitosan chloride (TMC).

In one embodiment, NO donors of the present invention comprise3-(2-Hydroxy-1-(1-methylethyl)-2-nitrosohydrazino)-1-propanamine,N-ethyl-2-(1-ethyl-hydroxy-2-nitrosohydrazino)-ethanamine, orS-Nitroso-N-acetylpenicillamine

In another embodiment, the bile acid is cholic acid. In anotherembodiment, the bile acid is chenodeoxycholic acid. In anotherembodiment, the bile acid is taurocholic acid. In another embodiment,the bile acid is taurochenodeoxycholic acid. In another embodiment, thebile acid is glycocholic acid. In another embodiment, the bile acid isglycochenocholic acid. In another embodiment, the bile acid is 3beta-monohydroxychloric acid. In another embodiment, the bile acid islithocholic acid. In another embodiment, the bile acid is 5beta-cholanic acid. In another embodiment, the bile acid is3,12-diol-7-one-5 beta-cholanic acid. In another embodiment, the bileacid is 3 alpha-hydroxy-12-ketocholic acid. In another embodiment, thebile acid is 3 beta-hydroxy-12-ketocholic acid. In another embodiment,the bile acid is 12 alpha-3 beta-dihydrocholic acid. In anotherembodiment, the bile acid is ursodesoxycholic acid.

In one embodiment, the enhancer is a nonionic surfactant. In oneembodiment, the enhancer is a nonionic polyoxyethylene ether surfaceactive agent (e.g one having an HLB value of 6 to 19, wherein theaverage number of polyoxyethylene units is 4 to 30). In anotherembodiment, the enhancer is an anionic surface active agents. In anotherembodiment, the enhancer is a cationic surface active agent. In anotherembodiment, the enhancer is an ampholytic surface active agent. In oneembodiment, zwitteruionic surfactants such as acylcarnitines serve asabsorption enhancers.

In another embodiment, the absorption enhancer is an effective oralabsorption enhancer for macromolecular drugs.In another embodiment, theabsorption enhancer is very water soluble.In another embodiment, theabsorption enhancer is fully, i. e. greater than 85%, absorbed by thegastro-intestinal tract. In another embodiment, the absorption enhanceris a coarse form. In another embodiment, the absorption enhancer ismicronized. In another embodiment, the absorption enhancer is amorphous.In another embodiment, the absorption enhancer isN-(5-chlorosalicyloyl)-8-aminocaprylic acid (5-CNAC). In anotherembodiment, the absorption enhancer is N-(10[2-hydroxybenzoyl]amino)decanoic acid (SNAD). In another embodiment, the absorption enhancer isN-(8- [2-hydroxybenzoyl]amino) caprylic acid (SNAC). In anotherembodiment, the absorption enhancer is CNAC, SNAD, SNAC, a monosodiumand/or disodium salts thereof, ethanol solvates of sodium salts thereofand the monohydrates of sodium salts thereof and any combinationsthereof. In another embodiment, the absorption enhancer is8-(N-2-hydroxy-4-methoxybenzoyl)-aminocaprylic acid (4-MOAC) andpharmaceutically acceptable salts thereof and/or amorphous andpolymorphic forms of 4-MOAC. In another embodiment, the absorptionenhancer is N-(8-[2-hydroxy-5-chlorobenzoyl]-amino)octanoic acid (alsoknown as 8-(N-2-hydroxy-5-chlorobenzoyDaminocaprylic acid)) (5-CNAC) andpharmaceutically acceptable salts thereof and/or amorphous andpolymorphic forms of 5-CNAC. In another embodiment, the absorptionenhancer is 4-[(2-hydroxy-4- chlorobenzoyl)amino]butanoate (also knownas 4-[(4-chloro-2-hydroxy- benzoyl)amino]butanoic acid) (4-CNAB) andpharmaceutically acceptable salts thereof, including its monosodium saltand/or amorphous and polymorphic forms of 4-CNAB.

In another embodiment, there is a synergistic effect in lowering bloodglucose level of SNAD and a protease inhibitor of the invention.Inanother embodiment, there is a synergistic effect in lowering bloodglucose level of SNAC and a protease inhibitor of the invention.

In another embodiment, the pharmaceutical compositions of the presentinvention comprise a delivery effective amount of one or more of theabsorption enhancers.In another embodiment, the pharmaceuticalcompositions of the present invention comprise an amount sufficient todeliver the active agent for the desired effect.

In another embodiment, the pharmaceutical compositions of the presentinvention comprise an amount of 2.5% to 99.4% by weight of an absorptionenhancer. In another embodiment, the pharmaceutical compositions of thepresent invention comprise an amount of 2.5% to 10% by weight of aabsorption enhancer. In another embodiment, the pharmaceuticalcompositions of the present invention comprise an amount of 8% to 15% byweight of a absorption enhancer. In another embodiment, thepharmaceutical compositions of the present invention comprise an amountof 10% to 20% by weight of a absorption enhancer. In another embodiment,the pharmaceutical compositions of the present invention comprise anamount of 15% to 30% by weight of an absorption enhancer. In anotherembodiment, the pharmaceutical compositions of the present inventioncomprise an amount of 20% to 40% by weight of an absorption enhancer. Inanother embodiment, the pharmaceutical compositions of the presentinvention comprise an amount of 30% to 50% by weight of an absorptionenhancer. In another embodiment, the pharmaceutical compositions of thepresent invention comprise an amount of 40% to 60% by weight of anabsorption enhancer. In another embodiment, the pharmaceuticalcompositions of the present invention comprise an amount of 50% to 70%by weight of an absorption enhancer. In another embodiment, thepharmaceutical compositions of the present invention comprise an amountof 70% to 99.4% by weight of an absorption enhancer. In anotherembodiment, the amount of an absorption enhancer in the presentcomposition is a delivery effective amount and can be determined for anyparticular carrier or biologically or chemically active agent by methodsknown to those skilled in the art.

In another embodiment, an absorption enhancer is a peptide. In anotherembodiment, an absorption enhancer is an amino acid. In anotherembodiment, a absorption enhancer is derived from amino acids.

In another embodiment, the amount of enhancer utilized in methods andcompositions of the present invention is 0.1 mg/dosage unit. In anotherembodiment, the amount of enhancer is 0.2 mg/dosage unit. In anotherembodiment, the amount is 0.3 mg/dosage unit. In another embodiment, theamount is 0.4 mg/dosage unit. In another embodiment, the amount is 0.6mg/dosage unit. In another embodiment, the amount is 0.8 mg/dosage unit.In another embodiment, the amount is 1 mg/dosage unit. In anotherembodiment, the amount is 1.5 mg/dosage unit. In another embodiment, theamount is 2 mg/dosage unit. In another embodiment, the amount is 2.5mg/dosage unit. In another embodiment, the amount is 3 mg/dosage unit.In another embodiment, the amount is 5 mg/dosage unit. In anotherembodiment, the amount is 7 mg/dosage unit. In another embodiment, theamount is 10 mg/dosage unit. In another embodiment, the amount is 12mg/dosage unit. In another embodiment, the amount is 15 mg/dosage unit.In another embodiment, the amount is 20 mg/dosage unit. In anotherembodiment, the amount is 30 mg/dosage unit. In another embodiment, theamount is 50 mg/dosage unit. In another embodiment, the amount is 70mg/dosage unit. In another embodiment, the amount is 100 mg/dosage unit.

In another embodiment, the amount of enhancer is 0.1-1 mg/dosage unit.In another embodiment, the amount of enhancer is 0.2-1 mg/dosage unit.In another embodiment, the amount is 0.3-1 mg/dosage unit. In anotherembodiment, the amount is 0.5-1 mg/dosage unit. In another embodiment,the amount is 0.1-2 mg/dosage unit. In another embodiment, the amount is0.2-2 mg/dosage unit. In another embodiment, the amount is 0.3-2mg/dosage unit. In another embodiment, the amount is 0.5-2 mg/dosageunit. In another embodiment, the amount is 1-2 mg/dosage unit. Inanother embodiment, the amount is 1-10 mg/dosage unit. In anotherembodiment, the amount is 2-10 mg/dosage unit. In another embodiment,the amount is 3-10 mg/dosage unit. In another embodiment, the amount is5-10 mg/dosage unit. In another embodiment, the amount is 1-20 mg/dosageunit. In another embodiment, the amount is 2-20 mg/dosage unit. Inanother embodiment, the amount is 3-20 mg/dosage unit. In anotherembodiment, the amount is 5-20 mg/dosage unit. In another embodiment,the amount is 10-20 mg/dosage unit. In another embodiment, the amount is10-100 mg/dosage unit. In another embodiment, the amount is 20-100mg/dosage unit. In another embodiment, the amount is 30-100 mg/dosageunit. In another embodiment, the amount is 50-100 mg/dosage unit. Inanother embodiment, the amount is 10-200 mg/dosage unit. In anotherembodiment, the amount is 20-200 mg/dosage unit. In another embodiment,the amount is 30-200 mg/dosage unit. In another embodiment, the amountis 50-200 mg/dosage unit. In another embodiment, the amount is 100-200mg/dosage unit. Each type and amount of enhancer represents a separateembodiment of the present invention.

In some embodiments, omega-3 fatty acid can be found in vegetablesources such as the seeds of chia, perilla, flax, walnuts, purslane,lingonberry, seabuckthorn, and hemp. In some embodiments, omega-3 fattyacids can also be found in the fruit of the acai palm. In anotherembodiment, the omega-3 fatty acid has been provided in the form of asynthetic omega-3 fatty acid. In one embodiment, the omega-3 fatty acidof methods and compositions of the present invention has been providedto the composition in the form of fish oil. In another embodiment, theomega-3 fatty acid has been provided in the form of canola oil. Inanother embodiment, the omega-3 fatty acid has been provided in the formof flaxseed oil. In another embodiment, the omega-3 fatty acid has beenprovided in the form of any other omega-3 fatty acid-rich source knownin the art. In another embodiment, the omega-3 fatty acid has beenprovided in the form of a synthetic omega-3 fatty acid. Each form ofomega-3 fatty acids represents a separate embodiment of the presentinvention.

In another embodiment, the omega-3 fatty acid of methods andcompositions of the present invention is an omega-3 polyunsaturatedfatty acid. In another embodiment, the omega-3 fatty acid is DHA, anomega-3, polyunsaturated, 22-carbon fatty acid also referred to as 4, 7,10, 13, 16, 19-docosahexaenoic acid. In another embodiment, the omega-3fatty acid is -linolenic acid (9, 12, 15-octadecatrienoic acid). Inanother embodiment, the omega-3 fatty acid is stearidonic acid (6, 9,12, 15-octadecatetraenoic acid). In another embodiment, the omega-3fatty acid is eicosatrienoic acid (ETA; 11, 14, 17-eicosatrienoic acid).In another embodiment, the omega-3 fatty acid is eicsoatetraenoic acid(8, 11, 14, 17-eicosatetraenoic acid). In one embodiment, the omega-3fatty acid is eicosapentaenoic acid (EPA; 5, 8, 11, 14,17-eicosapentaenoic acid). In another embodiment, the omega-3 fatty acidis eicosahexaenoic acid (also referred to as “EPA”; 5, 7, 9, 11, 14,17-eicosahexaenoic acid). In another embodiment, the omega-3 fatty acidis docosapentaenoic acid (DPA; 7, 10, 13, 16, 19-docosapenatenoic acid).In another embodiment, the omega-3 fatty acid is tetracosahexaenoic acid(6, 9, 12, 15, 18, 21-tetracosahexaenoic acid). In another embodiment,the omega-3 fatty acid is any other omega-3 fatty acid known in the art.Each omega-3 fatty acid represents a separate embodiment of the presentinvention.

In another embodiment, compositions of the present invention furthercomprise a coating that inhibits digestion of the composition in asubject's stomach. In one embodiment, coating inhibits digestion of thecomposition in a subject's stomach. In one embodiment, the coated dosageforms of the present invention release drug when pH move towardsalkaline range. In one embodiment, coating is a monolayer, wherein inother embodiments coating applied in multilayers. In one embodiment,coating is a bioadhesive polymer that selectively binds the intestinalmucosa and thus enables drug release in the attachment site. In oneembodiment, the enteric coating is an enteric film coating. In someembodiment, coating comprises biodegradable polysaccharide, chitosan,aquateric aqueous, aquacoat ECD, azo polymer, cellulose acetatephthalate, cellulose acetate trimelliate, hydroxypropylmethyl cellulosephthalate, gelatin, poly vinyl acetate phthalate, hydrogel, pulsincap,or a combination thereof. In one embodiment, pH sensitive coating willbe used according to the desired release site and/or profile as known toone skilled in the art.

In one embodiment, the coating is an enteric coating. Methods forenteric coating are well known in the art, and are described, forexample, in Siepmann F, Siepmann J et al, Blends of aqueous polymerdispersions used for pellet coating: importance of the particle size. JControl Release 2005; 105(3): 226-39; and Huyghebaert N, Vermeire A,Remon JP. In vitro evaluation of coating polymers for enteric coatingand human ileal targeting. Int J Pharm 2005; 298(1): 26-37. Each methodrepresents a separate embodiment of the present invention.

In another embodiment, Eudragit®, an acrylic polymer, is used as theenteric coating. The use of acrylic polymers for the coating ofpharmaceutical preparations is well known in the art. Eudragit AcrylicPolymers have been shown to be safe, and are neither absorbed normetabolized by the body, but rather are eliminated.

In another embodiment, the coating is a gelatin coating. In anotherembodiment, microencapsulation is used to protect the insulin againstdecomposition in the stomach. In another embodiment, the coating is agelatin coating. In another embodiment, microencapsulation is used toprotect Exenatide against decomposition in the stomach. Methods forapplying a gelatin coating and for microencapsulation are well known inthe art. Each method represents a separate embodiment of the presentinvention.

In another embodiment, the coating is a film-coating. In anotherembodiment, the coating is ethylcellulose. In another embodiment, thecoating is a water-based dispersion of ethylcellulose, e.g.hydroxypropylmethylcelullose (HPMC) E15. In another embodiment, thecoating is a gastro-resistant coating, e.g. a polymer containingcarboxylic acid groups as a functional moiety. In another embodiment,the coating is a monolithic matrix. In another embodiment, the coatingis cellulose ether (e.g. hypromellose (HPMC). Each type of coatingrepresents a separate embodiment of the present invention.

In another embodiment, the present invention provides a compositioncomprising an active protein of the invention, a protease inhibitor, andan absorption enhancer. In another embodiment, the present inventionprovides a composition comprising an active protein of the invention, aprotease inhibitor, an omega-3 fatty acid, and an absorption enhancer.In another embodiment, the present invention provides a compositioncomprising an active protein of the invention, a protease inhibitor, anomega-3 fatty acid, EDTA or Na-EDTA, and an absorption enhancer.

In another embodiment, the present invention provides that the use of atleast one protease inhibitor and an absorption enhancer in a single oralcomposition dramatically, unexpectedly, increase the bioavailability ofa protein of the invention.In another embodiment, the present inventionprovides that the use of at least one protease inhibitor and anabsorption enhancer in a single oral composition dramatically,unexpectedly, increase the bioavailability of insulin.In anotherembodiment, the present invention provides that the use of at least oneprotease inhibitor and an absorption enhancer in a single oralcomposition dramatically, unexpectedly, increase the bioavailability ofExenatide.

In another embodiment, the present invention provides that the use of atleast one protease inhibitor and an absorption enhancer in a single oralcomposition dramatically, unexpectedly, increase the bioavailability ofa protein of the invention.In another embodiment, the present inventionprovides that the use of at least one protease inhibitor and anabsorption enhancer in a single oral composition dramatically,unexpectedly, increase the bioavailability of insulin.In anotherembodiment, the present invention provides that the use of at least oneprotease inhibitor and an absorption enhancer in a single oralcomposition dramatically, unexpectedly, increase the bioavailability ofExenatide.

In another embodiment, the present invention provides that the use of atleast one protease inhibitor and SNAC in a single oral compositiondramatically, unexpectedly, increase the bioavailability of a protein ofthe invention.In another embodiment, the present invention provides thatthe use of at least one protease inhibitor and SNAC in a single oralcomposition dramatically, unexpectedly, increase the bioavailability ofinsulin.In another embodiment, the present invention provides that theuse of at least one protease inhibitor and SNAC in a single oralcomposition dramatically, unexpectedly, increase the bioavailability ofExenatide.

In another embodiment, the present invention provides that the use of atleast one protease inhibitor and SNAD in a single oral compositiondramatically, unexpectedly, increase the bioavailability of a protein ofthe invention.In another embodiment, the present invention provides thatthe use of at least one protease inhibitor and SNAD in a single oralcomposition dramatically, unexpectedly, increase the bioavailability ofinsulin.In another embodiment, the present invention provides that theuse of at least one protease inhibitor and SNAD in a single oralcomposition dramatically, unexpectedly, increase the bioavailability ofExenatide.

In another embodiment, the present invention provides that the use of aserpin and SNAC or SNAD in a single oral composition dramatically,unexpectedly, increase the bioavailability of a protein of theinvention.In another embodiment, the present invention provides that theuse of a serpin and SNAC or SNAD in a single oral compositiondramatically, unexpectedly, increase the bioavailability of insulin.Inanother embodiment, the present invention provides that the use of aserpin and SNAC or SNAD in a single oral composition dramatically,unexpectedly, increase the bioavailability of Exenatide. In anotherembodiment, the present invention provides that the use of a serpin andSNAC or SNAD in a single oral composition dramatically, unexpectedly,increase the bioavailability of a protein of the invention.

In another embodiment, the present invention provides that the use of atrypsin inhibitor and SNAC or SNAD in a single oral compositiondramatically, unexpectedly, increase the bioavailability of insulin.Inanother embodiment, the present invention provides that the use of atrypsin inhibitor and SNAC or SNAD in a single oral compositiondramatically, unexpectedly, increase the bioavailability of Exenatide.In another embodiment, the present invention provides that the use ofSBTI or Aprotinin and SNAC or SNAD in a single oral compositiondramatically, unexpectedly, increase the bioavailability of a protein ofthe invention. In another embodiment, the present invention providesthat the use of SBTI or Aprotinin and SNAC or SNAD in a single oralcomposition dramatically, unexpectedly, increase the bio availability ofinsulin. In another embodiment, the present invention provides that theuse of SBTI or Aprotinin and SNAC or SNAD in a single oral compositiondramatically, unexpectedly, increase the bio availability of Exenatide.

In another embodiment, the present invention provides that the use of aCysteine protease inhibitor and SNAC or SNAD in a single oralcomposition dramatically, unexpectedly, increase the bioavailability ofa protein of the invention.In another embodiment, the present inventionprovides that the use of a a Cysteine protease inhibitor and SNAC orSNAD in a single oral composition dramatically, unexpectedly, increasethe bioavailability of insulin.In another embodiment, the presentinvention provides that the use of a Cysteine protease inhibitor andSNAC or SNAD in a single oral composition dramatically, unexpectedly,increase the bioavailability of Exenatide.

In another embodiment, the present invention provides that the use of aThreonine protease inhibitor and SNAC or SNAD in a single oralcomposition dramatically, unexpectedly, increase the bio availability ofa protein of the invention.In another embodiment, the present inventionprovides that the use of a Threonine protease inhibitor and SNAC or SNADin a single oral composition dramatically, unexpectedly, increase thebioavailability of insulin. In another embodiment, the present inventionprovides that the use of a Threonine protease inhibitor and SNAC or SNADin a single oral composition dramatically, unexpectedly, increase thebio availability of Exenatide.

In another embodiment, the present invention provides that the use of aMetalloprotease protease inhibitor and SNAC or SNAD in a single oralcomposition dramatically, unexpectedly, increase the bioavailability ofa protein of the invention. In another embodiment, the present inventionprovides that the use of a Metalloprotease protease inhibitor and SNACor SNAD in a single oral composition dramatically, unexpectedly,increase the bioavailability of insulin. In another embodiment, thepresent invention provides that the use of a Metalloprotease proteaseinhibitor and SNAC or SNAD in a single oral composition dramatically,unexpectedly, increase the bio availability of Exenatide.

In another embodiment, the present invention provides that the use of anAspartic protease inhibitor and SNAC or SNAD in a single oralcomposition dramatically, unexpectedly, increase the bioavailability ofa protein of the invention. In another embodiment, the present inventionprovides that the use of an Aspartic protease inhibitor and SNAC or SNADin a single oral composition dramatically, unexpectedly, increase thebioavailability of insulin. In another embodiment, the present inventionprovides that the use of an Aspartic protease inhibitor and SNAC or SNADin a single oral composition dramatically, unexpectedly, increase thebioavailability of Exenatide.

In another embodiment, the present invention provides that the use of aprotease inhibitor and SNAC or SNAD in a single oral compositiondramatically, unexpectedly, increase the bioavailability of insulin in ahuman subject by at least 10%. In another embodiment, the presentinvention provides that the use of a protease inhibitor and SNAC or SNADin a single oral composition dramatically, unexpectedly, increase thebioavailability of Exenatide in a human subject by at least 10%. Inanother embodiment, the present invention provides that the use of aprotease inhibitor and SNAC or SNAD in a single oral compositiondramatically, unexpectedly, increase the bioavailability of insulin in ahuman subject by at least 20%. In another embodiment, the presentinvention provides that the use of a protease inhibitor and SNAC or SNADin a single oral composition dramatically, unexpectedly, increase thebioavailability of Exenatide in a human subject by at least 20%. Inanother embodiment, the present invention provides that the use of aprotease inhibitor and SNAC or SNAD in a single oral compositiondramatically, unexpectedly, increase the bioavailability of insulin in ahuman subject by at least 30%. In another embodiment, the presentinvention provides that the use of a protease inhibitor and SNAC or SNADin a single oral composition dramatically, unexpectedly, increase thebioavailability of Exenatide in a human subject by at least 30%. Inanother embodiment, the present invention provides that the use of aprotease inhibitor and SNAC or SNAD in a single oral compositiondramatically, unexpectedly, increase the bioavailability of insulin in ahuman subject by at least 40%. In another embodiment, the presentinvention provides that the use of a protease inhibitor and SNAC or SNADin a single oral composition dramatically, unexpectedly, increase thebioavailability of Exenatide in a human subject by at least 40%. Inanother embodiment, the present invention provides that the use of aprotease inhibitor and SNAC or SNAD in a single oral compositiondramatically, unexpectedly, increase the bioavailability of insulin in ahuman subject by at least 50%. In another embodiment, the presentinvention provides that the use of a protease inhibitor and SNAC or SNADin a single oral composition dramatically, unexpectedly, increase thebio availability of Exenatide in a human subject by at least 50%.Inanother embodiment, the present invention provides that the use of aprotease inhibitor and SNAC or SNAD in a single oral compositiondramatically, unexpectedly, increase the bioavailability of insulin in ahuman subject by at least 60%.In another embodiment, the presentinvention provides that the use of a protease inhibitor and SNAC or SNADin a single oral composition dramatically, unexpectedly, increase thebioavailability of Exenatide in a human subject by at least 60%. Inanother embodiment, the present invention provides that the use of aprotease inhibitor and SNAC or SNAD in a single oral compositiondramatically, unexpectedly, increase the bioavailability of insulin in ahuman subject by at least 70%. In another embodiment, the presentinvention provides that the use of a protease inhibitor and SNAG or SNADin a single oral composition dramatically, unexpectedly, increase thebioavailability of Exenatide in a human subject by at least 70%. Inanother embodiment, the present invention provides that the use of aprotease inhibitor and SNAC or SNAD in a single oral compositiondramatically, unexpectedly, increase the bioavailability of insulin in ahuman subject by at least 80%. In another embodiment, the presentinvention provides that the use of a protease inhibitor and SNAC or SNADin a single oral composition dramatically, unexpectedly, increase thebioavailability of Exenatide in a human subject by at least 80%. Inanother embodiment, the present invention provides that the use of aprotease inhibitor and SNAC or SNAD in a single oral compositiondramatically, unexpectedly, increase the bioavailability of insulin in ahuman subject by at least 90%. In another embodiment, the presentinvention provides that the use of a protease inhibitor and SNAC or SNADin a single oral composition dramatically, unexpectedly, increase thebioavailability of Exenatide in a human subject by at least 90%. Inanother embodiment, the present invention provides that the use of aprotease inhibitor and SNAC or SNAD in a single oral compositiondramatically, unexpectedly, increase the bioavailability of insulin in ahuman subject by at least 100%. In another embodiment, the presentinvention provides that the use of a protease inhibitor and SNAC or SNADin a single oral composition dramatically, unexpectedly, increase thebioavailability of Exenatide in a human subject by at least 100%.

In another embodiment, the present invention provides that the use ofAprotinin or SBTI and SNAC or SNAD in a single oral compositiondramatically, unexpectedly, increase the bioavailability of insulin in ahuman subject by at least 10%.In another embodiment, the presentinvention provides that the use of Aprotinin or SBTI and SNAC or SNAD ina single oral composition dramatically, unexpectedly, increase thebioavailability of insulin in a human subject by at least 20%. Inanother embodiment, the present invention provides that the use ofAprotinin or SBTI and SNAC or SNAD in a single oral compositiondramatically, unexpectedly, increase the bioavailability of insulin in ahuman subject by at least 30%.In another embodiment, the presentinvention provides that the use of Aprotinin or SBTI and SNAC or SNAD ina single oral composition dramatically, unexpectedly, increase thebioavailability of insulin in a human subject by at least 40%.In anotherembodiment, the present invention provides that the use of Aprotinin orSBTI and SNAC or SNAD in a single oral composition dramatically,unexpectedly, increase the bioavailability of insulin in a human subjectby at least 50%.In another embodiment, the present invention providesthat the use of Aprotinin or SBTI and SNAC or SNAD in a single oralcomposition dramatically, unexpectedly, increase the bioavailability ofinsulin in a human subject by at least 60%.In another embodiment, thepresent invention provides that the use of Aprotinin or SBTI and SNAC orSNAD in a single oral composition dramatically, unexpectedly, increasethe bioavailability of insulin in a human subject by at least 70%. Inanother embodiment, the present invention provides that the use ofAprotinin or SBTI and SNAG or SNAD in a single oral compositiondramatically, unexpectedly, increase the bioavailability of insulin in ahuman subject by at least 80%. In another embodiment, the presentinvention provides that the use of Aprotinin or SBTI and SNAC or SNAD ina single oral composition dramatically, unexpectedly, increase thebioavailability of insulin in a human subject by at least 90%. Inanother embodiment, the present invention provides that the use ofAprotinin or SBTI and SNAC or SNAD in a single oral compositiondramatically, unexpectedly, increase the bioavailability of insulin in ahuman subject by at least 100%.

In another embodiment, this invention further provides the use ofsustained release dosage forms (e.g. sustained releasemicroencapsulation) that enable the treatment frequency to be reduced toonce or twice a day. In another embodiment, the insulin dosage isincreased correspondingly with decreasing frequency of administration.In another embodiment, the Exenatide dosage is increased correspondinglywith decreasing frequency of administration. Each type of coating,absorption enhancer, dosage form, etc, that inhibits digestion of thecomposition in the stomach represents a separate embodiment of thepresent invention.

Methods of measuring insulin levels are well known in the art. In oneembodiment, levels of recombinant insulin are measuring using a humaninsulin radio-immunoassay (RIA) kit, e.g. the kit manufactured by LincoResearch Inc, (St. Charles, Mo.). In another embodiment, levels of Cpeptide are measured as well, to determine the relative contributions ofendogenous and exogenous insulin to observed rises in insulin levels. Inanother embodiment, insulin ELISA kits are used. In another embodiment,insulin levels are measured by any other method known in the art. Inanother embodiment, Exenatide levels are measured by a method known inthe art. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, a multiparticulate dosage forms is used toinhibit digestion of the composition in the stomach. In anotherembodiment, a multiparticulate dosage forms is used to inhibit digestionof the composition in the stomach.

In another embodiment, the present invention provides a method for oraladministration of a protein with an enzymatic activity to a subject,whereby a substantial fraction of the protein retains the enzymaticactivity after absorption through an intestinal mucosal barrier of thesubject, comprising administering orally to the subject a pharmaceuticalcomposition comprising the protein, a protease inhibitor, and anabsorption enhancer, thereby orally administering a protein with anenzymatic activity to a subject.

In another embodiment, the present invention provides a method for oraladministration of a protein with an enzymatic activity to a subject,whereby a substantial fraction of the protein retains the enzymaticactivity after absorption through an intestinal mucosal barrier of thesubject, comprising administering orally to the subject a pharmaceuticalcomposition comprising the protein, a protease inhibitor, and SNAC orSNAD, thereby orally administering a protein with an enzymatic activityto a subject.

In another embodiment, the present invention provides a method for oraladministration of insulin to a subject, whereby a substantial fractionof insulin retains its activity after absorption through an intestinalmucosal barrier of the subject, comprising administering orally to thesubject a pharmaceutical composition comprising insulin, a proteaseinhibitor, and SNAC or SNAD, thereby orally administering a protein withan enzymatic activity to a subject. In another embodiment, the presentinvention provides a method for oral administration of Exenatide to asubject, whereby a substantial fraction of Exenatide retains itsactivity after absorption through an intestinal mucosal barrier of thesubject, comprising administering orally to the subject a pharmaceuticalcomposition comprising Exenatide at least one protease inhibitor, andSNAC or SNAD, thereby orally administering a protein with an enzymaticactivity to a subject.

In another embodiment, the present invention provides a method for oraladministration of a protein with an enzymatic activity to a subject,whereby a substantial fraction of the protein retains the enzymaticactivity after absorption through an intestinal mucosal barrier of thesubject, comprising administering orally to the subject a pharmaceuticalcomposition comprising the protein, at least one protease inhibitor,SNAC or SNAD, and an omega-3 fatty acid, thereby orally administering aprotein with an enzymatic activity to a subject. In another embodiment,the present invention provides a method for oral administration of aprotein with an enzymatic activity to a subject, whereby a substantialfraction of the protein retains the enzymatic activity after absorptionthrough an intestinal mucosal barrier of the subject, comprisingadministering orally to the subject a pharmaceutical compositioncomprising the protein, at least one protease inhibitor, and SNAC orSNAD, EDTA (or a salt thereof), and an omega-3 fatty acid, therebyorally administering a protein with an enzymatic activity to a humansubject.

In another embodiment, the present invention provides a method for oraladministration of insulin to a subject, whereby a substantial fractionof insulin retains its activity after absorption through an intestinalmucosal barrier of the subject, comprising administering orally to thesubject a pharmaceutical composition comprising insulin, at least oneprotease inhibitor, SNAC or SNAD, an omega-3 fatty acid, and Na-EDTA,thereby orally administering a protein with an enzymatic activity to asubject. In another embodiment, the present invention provides a methodfor oral administration of Exenatide to a subject, whereby a substantialfraction of Exenatide retains its activity after absorption through anintestinal mucosal barrier of the subject, comprising administeringorally to the subject a pharmaceutical composition comprising Exenatide,at least one protease inhibitor, SNAC or SNAD,an omega-3 fatty acid, andNa-EDTA, thereby orally administering a protein with an enzymaticactivity to a subject.

In another embodiment, the present invention provides a method fortreating diabetes mellitus in a human subject, comprising administeringorally to the subject a pharmaceutical composition comprising aninsulin, at least one protease inhibitor, and SNAC or SNAD, therebytreating diabetes mellitus. In another embodiment, the present inventionprovides a method for treating diabetes mellitus in a human subject,comprising administering orally to the subject a pharmaceuticalcomposition comprising an insulin and at least one protease inhibitor,and SNAC or SNAD, and omega-3 fatty acid thereby treating diabetesmellitus. In another embodiment, the present invention provides a methodfor treating diabetes mellitus in a human subject, comprisingadministering orally to the subject a pharmaceutical compositioncomprising an insulin and at least one protease inhibitor, and SNAC orSNAD, EDTA (or a salt thereof), and omega-3 fatty acid thereby treatingdiabetes mellitus.

In another embodiment, the present invention provides a method fortreating diabetes mellitus in a human subject, comprising administeringorally to the subject a pharmaceutical composition comprising anExenatide, at least one protease inhibitor, and SNAC or SNAD, therebytreating diabetes mellitus. In another embodiment, the present inventionprovides a method for treating diabetes mellitus in a human subject,comprising administering orally to the subject a pharmaceuticalcomposition comprising an Exenatide and at least one protease inhibitor,and SNAC or SNAD, and omega-3 fatty acid thereby treating diabetesmellitus. In another embodiment, the present invention provides a methodfor treating diabetes mellitus in a human subject, comprisingadministering orally to the subject a pharmaceutical compositioncomprising an Exenatide and at least one protease inhibitor, and SNAC orSNAD, EDTA (or a salt thereof), and omega-3 fatty acid thereby treatingdiabetes mellitus.

In one embodiment, the diabetes mellitus is Type I diabetes. In anotherembodiment, the diabetes mellitus is Type II diabetes. In anotherembodiment, the diabetes mellitus is insulin-dependent diabetes. Inanother embodiment, the diabetes mellitus is non-insulin-dependentdiabetes. In another embodiment, the diabetes mellitus is any other typeof diabetes known in the art. Each possibility represents a separateembodiment of the present invention.

In one embodiment, three treatments a day of the insulin composition areadministered. In another embodiment, two treatments a day areadministered. In another embodiment, four treatments a day areadministered. In another embodiment, one treatment a day isadministered. In another embodiment, more than four treatments a day areadministered. Each possibility represents a separate embodiment of thepresent invention.

Any of the methods of the present invention may utilize, in variousembodiments, any of the compositions of the present invention.

In another embodiment, the present invention provides a composition fororal administration of insulin, comprising an insulin protein at leastone protease inhibitor, and SNAG or SNAD, whereby a substantial fractionof the insulin retains the enzymatic activity after absorption throughan intestinal mucosal barrier of a human subject.

In one embodiment, the present invention provides the use of a protein,at least one protease inhibitor, and SNAC or SNAD in the manufacture ofa medicament for oral administration of a protein with an enzymaticactivity to a subject, whereby a substantial fraction of the proteinretains the enzymatic activity after absorption through an intestinalmucosal barrier of the subject. In one embodiment, the present inventionprovides the use of a protein, at least one protease inhibitor, SNAC orSNAD, and an omega-3 fatty acid in the manufacture of a medicament fororal administration of a protein with an enzymatic activity to asubject, whereby a substantial fraction of the protein retains theenzymatic activity after absorption through an intestinal mucosalbarrier of the subject. In one embodiment, the present inventionprovides the use of a protein, at least one protease inhibitor, SNAC orSNAD, Na-EDTA, and an omega-3 fatty acid in the manufacture of amedicament for oral administration of a protein with an enzymaticactivity to a subject, whereby a substantial fraction of the proteinretains the enzymatic activity after absorption through an intestinalmucosal barrier of the subject.

In one embodiment, the present invention provides the use of an insulinprotein, at least one protease inhibitor, and SNAC or SNAD in themanufacture of a medicament for treating diabetes mellitus in a subject.In one embodiment, the present invention provides the use of an insulinprotein, at least one protease inhibitor, SNAC or SNAD, and an omega-3fatty acid in the manufacture of a medicament for treating diabetesmellitus in a subject. In one embodiment, the present invention providesthe use of an insulin protein, at least one protease inhibitor, SNAC orSNAD, Na-EDTA, and an omega-3 fatty acid in the manufacture of amedicament for treating diabetes mellitus in a subject.

In one embodiment, the present invention provides the use of anExenatide protein, at least one protease inhibitor, and SNAC or SNAD inthe manufacture of a medicament for treating diabetes mellitus in asubject. In one embodiment, the present invention provides the use of anExenatide protein, at least one protease inhibitor, SNAC or SNAD, and anomega-3 fatty acid in the manufacture of a medicament for treatingdiabetes mellitus in a subject. In one embodiment, the present inventionprovides the use of an Exenatide protein, at least one proteaseinhibitor, SNAC or SNAD, Na-EDTA, and an omega-3 fatty acid in themanufacture of a medicament for treating diabetes mellitus in a subject.

In one embodiment, methods and compositions of the present inventionhave the advantage of more closely mimicking physiological insulinsecretion by the pancreas. When insulin is secreted into the portalvein, the liver is exposed to a greater insulin concentration thanperipheral tissues. Similarly, insulin administered according to thepresent invention reaches the intestine and is absorbed in the bodythrough the intestine and through the portal system to the liver. Thisabsorption route thus resembles the physiological secretion of insulinby the pancreas, enabling, in this embodiment, delicate control of theblood glucose level and the metabolic activities of the liver and theperipheral organs controlled by insulin. By contrast, when insulin isadministered to insulin-deficient diabetic patients via the peripheralvenous system, the concentration of insulin in the portal vein issimilar to that in the peripheral circulation, resulting inhypoinsulinemia in the portal vein and the liver and hyperinsulinemia inthe peripheral venous system. This leads, in one embodiment, to anabnormal pattern of glucose disposal.

In another embodiment, different constituents of compositions of thepresent composition are absorbed at different rates from the intestinallumen into the blood stream. The absorption of the bile acid, in oneembodiment, is significantly faster than the absorption of insulin.

For this reason, in another embodiment, a drug regimen involvingingestion of a pair of pills at spaced intervals, e.g., a second pillcontaining a higher concentration of enhancer is taken at a definedinterval (e.g. 30 minutes) after the first pill. In another embodiment,certain of the constituents are microencapsulated to enhance theabsorption of the insulin into the system. In another embodiment,certain of the constituents are microencapsulated to enhance theabsorption of the Exenatide into the system.

In one embodiment, a treatment protocol of the present invention istherapeutic. In another embodiment, the protocol is prophylactic. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, solid carriers/diluents for use in methods andcompositions of the present invention include, but are not limited to, agum, a starch (e.g. corn starch, pregeletanized starch), a sugar (e.g.,lactose, mannitol, sucrose, dextrose), a cellulosic material (e.g.microcrystalline cellulose), an acrylate (e.g. polymethylacrylate),calcium carbonate, magnesium oxide, talc, or mixtures thereof.

In another embodiment, the compositions further comprise binders (e.g.acacia, cornstarch, gelatin, carbomer, ethyl cellulose, guar gum,hydroxypropyl cellulose, hydroxypropyl methyl cellulose, povidone),disintegrating agents (e.g. cornstarch, potato starch, alginic acid,silicon dioxide, croscarmelose sodium, crospovidone, guar gum, sodiumstarch glycolate), buffers (e.g., Tris-HCl., acetate, phosphate) ofvarious pH and ionic strength, additives such as albumin or gelatin toprevent absorption to surfaces, detergents (e.g., Tween 20, Tween 80,Pluronic F68, bile acid salts), surfactants (e.g. sodium laurylsulfate), permeation enhancers, solubilizing agents (e.g., glycerol,polyethylene glycerol), anti-oxidants (e.g., ascorbic acid, sodiummetabisulfite, butylated hydroxyanisole), stabilizers (e.g.hydroxypropyl cellulose, hyroxypropylmethyl cellulose), viscosityincreasing agents(e.g. carbomer, colloidal silicon dioxide, ethylcellulose, guar gum), sweeteners (e.g. aspartame, citric acid),preservatives (e.g., Thimerosal, benzyl alcohol, parabens), lubricants(e.g. stearic acid, magnesium stearate, polyethylene glycol, sodiumlauryl sulfate), flow-aids (e.g. colloidal silicon dioxide),plasticizers (e.g. diethyl phthalate, triethyl citrate), emulsifiers(e.g. carbomer, hydroxypropyl cellulose, sodium lauryl sulfate), polymercoatings (e.g., poloxamers or poloxamines), coating and film formingagents (e.g. ethyl cellulose, acrylates, polymethacrylates) and/oradjuvants. Each of the above excipients represents a separate embodimentof the present invention.

In some embodiments, the dosage forms of the present invention areformulated to achieve an immediate release profile, an extended releaseprofile, or a delayed release profile. In some embodiments, the releaseprofile of the composition is determined by using specific excipientsthat serve for example as binders, disintegrants, fillers, or coatingmaterials. In one embodiment, the composition will be formulated toachieve a particular release profile as known to one skilled in the art.

In one embodiment, the composition is formulated as an oral dosage form.In one embodiment, the composition is a solid oral dosage formcomprising tablets, chewable tablets, or capsules. In one embodiment thecapsules are soft gelatin capsules. In another embodiment, capsules asdescribed herein are hard-shelled capsules.In another embodiment,capsules as described herein are soft-shelled capsules.In anotherembodiment, capsules as described herein are made from gelatine. Inanother embodiment, capsules as described herein are made fromplant-based gelling substances like carrageenans and modified forms ofstarch and cellulose.

In other embodiments, controlled- or sustained-release coatings utilizedin methods and compositions of the present invention include formulationin lipophilic depots (e.g. fatty acids, waxes, oils).

The compositions also include, in another embodiment, incorporation ofthe active material into or onto particulate preparations of polymericcompounds such as polylactic acid, polglycolic acid, hydrogels, etc, oronto liposomes, microemulsions, micelles, unilamellar or multilamellarvesicles, erythrocyte ghosts, or spheroplasts.) Such compositions willinfluence the physical state, solubility, stability, rate of in vivorelease, and rate of in vivo clearance. In another embodiment,particulate compositions of the active ingredients are coated withpolymers (e.g. poloxamers or poloxamines)

In another embodiment, the compositions containing the insulin andomega-3 fatty acid are delivered in a vesicle, e.g. a liposome (seeLanger, Science 249:1527-1533 (1990); Treat et al., in Liposomes in theTherapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler(eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp.317-327; see generally ibid). In another embodiment, the compositionscontaining the Exenatide and omega-3 fatty acid are delivered in avesicle, e.g. a liposome (see Langer, Science 249:1527-1533 (1990);Treat et al., in Liposomes in the Therapy of Infectious Disease andCancer, Lopez- Berestein and Fidler (eds.), Liss, New York, pp. 353-365(1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid).

The preparation of pharmaceutical compositions that contain an activecomponent, for example by mixing, granulating, or tablet-formingprocesses, is well understood in the art. The active therapeuticingredient is often mixed with excipients that are pharmaceuticallyacceptable and compatible with the active ingredient. For oraladministration, the active ingredients of compositions of the presentinvention are mixed with additives customary for this purpose, such asvehicles, stabilizers, or inert diluents, and converted by customarymethods into suitable forms for administration, such as tablets, coatedtablets, hard or soft gelatin capsules, aqueous, alcoholic or oilysolutions.

Each of the above additives, excipients, formulations and methods ofadministration represents a separate embodiment of the presentinvention.

In one embodiment, the term “treating” refers to curing a disease. Inanother embodiment, “treating” refers to preventing a disease. Inanother embodiment, “treating” refers to reducing the incidence of adisease. In another embodiment, “treating” refers to amelioratingsymptoms of a disease. In another embodiment, “treating” refers toinducing remission. In another embodiment, “treating” refers to slowingthe progression of a disease.

EXPERIMENTAL DETAILS SECTION Example 1 Insulin, SNAC, AND ProteaseInhibitors Oral Formulations Materials and Experimental MethodsFormulation

X [please provide] days prior to dosing, formulation were preparedcontaining: (1) 6 mg insulin, 250 mg SNAC, 125 mg SBTI in [what is theexact carrier], (2). 6 mg insulin, 250 mg SNAC, 2.5 mg Aprotinin, 125 mgSBTI in [what is the exact carrier], (3) 6 mg insulin, 250 mg SNAC, 2.5mg Aprotinin,125 mg SBTI in [what is the exact carrier]. (4) 6 mginsulin, 250 mg SNAC in [what is the exact carrier]. The formulationswere stored in the refrigerator (4° C.) until dosing.

Results

In the next experiment, the formulations as describes in the“Formulation” section were orally consumed by 3 healthy human subjects.As shown in FIG. 1, blood glucose levels were significantly reduced andmore stable in human subjects treated with formulations (1) and (3).These results suggest that formulations comprising a combination of aprotease inhibitor and SNAC are superior to formulations comprising onlySNAC. Specifically, the combination of SNAC and SBTI is superior to anyother formulation used. It is important to notice that the effect ofSNAG and a protease inhibitor is synergistic because formulationsconsisting insulin and a single protease inhibitor do not have a bloodglucose lowering effect- no differences in the reduction of bloodglucose levels were observed in previous experiment with the sameformulations comprising a single protease inhibitor (SBTI or Aprotinin).Thus, the results regarding the reduction in blood glucose levels withinsulin and a combination of SNAC and at least one protease inhibitorwas unexpected. The formulation comprising SNAC and SBTI had asynergistic, utmost effect in lowering blood glucose levels

As shown in FIG. 2, blood C-peptide levels were significantly reduced inhuman subjects treated with formulation (1) and (3). These results alsosuggest that the combination of SNAC and a protease inhibitor such asSBTI had a synergistic effect in lowering blood C-peptide levels.

It should be emphasized that no reduction of C-peptide levels wereobserved in a previous experiment wherein formulations comprising asingle protease inhibitor (SBTI or Aprotinin) were used. Thus, theresults regarding the reduction in blood C-peptide levels with insulinand a combination of a protease inhibitor such as SBTI and SNAG wasunexpected.

Example 2 Optimization of Source of Omega-3 Fatty Acids

Various omega-3 fatty acids or sources of omega-3 fatty acids (e.g.those listed above in the specification) are compared for their abilityto preserve insulin following oral administration in methods andcompositions of the present invention. Insulin tablets or capsules areformulated as described in the above Examples, except that the insulinis dissolved in the alternate source instead of in fish oil. The mosteffective source of omega-3 fatty acids is used in subsequent Examples.

Example 3 Optimization of a Protease Inhibitor

Various protease inhibitors (either non-toxic or having an acceptabletoxicity profile; e.g. those listed above in the specification) arecompared for their ability to preserve insulin following oraladministration in methods and compositions of the present invention.Insulin and/or Exenatide tablets or capsules are formulated as describedin the above Examples, except that the alternate protease inhibitors aresubstituted for SBTI and/or Aprotinin. Amounts of the proteaseinhibitors are also varied, to determine the optimal amounts. The mosteffective protease inhibitor/amount is used in subsequent Examples.

Example 4 Optimization of an Enhancer

Various enhancers (e.g. those listed above in the specification) arecompared for their ability to facilitate absorption of insulin followingoral administration in methods and compositions of the presentinvention. Insulin tablets or capsules are formulated as described inthe above Examples, except that the alternate enhancers are substitutedfor EDTA. Amounts of the enhancers are also varied, to determine theoptimal amounts. The most effective enhancer/amount is used insubsequent experiments.

Example 5 Optimization of Type and Amount of Insulin

Various types and amounts of insulin e.g. those listed above in thespecification) are compared for their ability to regulate blood sugar inmethods and compositions of the present invention. Insulin tablets orcapsules are formulated as described in the above Examples, except thatthe type and amount of insulin is varied. The most effective type/amountof insulin is used in clinical trials.

1-31. (canceled)
 32. An oral pharmaceutical composition comprising aprotein having a molecular weight of up to 100,000 Daltons; a proteaseinhibitor; and a compound selected from the group consisting ofN-(8-[2-hydroxybenzoyl]amino)caprylate (SNAC),N-(10-[2-hydroxybenzoyl]amino)decanoate (SNAD), a salt of said SNAC orsaid SNAD, and a combination thereof.
 33. The composition of claim 32,wherein said protein is a recombinant protein.
 34. The composition ofclaim 32, wherein said enhancer is a salt of said SNAC or said SNAD, andsaid salt is selected from the group consisting of a monosodium salt, adisodium salt, and a combination thereof.
 35. The composition of claim32, wherein said protein is selected from the group consisting ofinsulin, a glucagon, an interferon gamma, an interferon alpha, a growthhormone, an erythropoietin, a GLP-1, a GLP-1 analogue, and granulocytecolony stimulating factor (G-CSF).
 36. The composition of claim 32,wherein said protease inhibitor is selected from the group consisting ofa serpin, a suicide inhibitor, a transition state inhibitor, a proteinprotease inhibitor, a chelating agent, a Cysteine protease inhibitor, aThreonine protease inhibitor, an Aspartic protease inhibitor, and aMetalloprotease inhibitor.
 37. The composition of claim 36, wherein saidprotease inhibitor is a serpin, and said serpin is a trypsin inhibitor.38. The composition of claim 32, wherein said protease inhibitor isselected from the group consisting of Lima bean trypsin inhibitor,Aprotinin, soy bean trypsin inhibitor (SBTI), and Ovomucoid.
 39. Thecomposition of claim 32, further comprising an omega-3 fatty acid. 40.The composition of claim 32, further comprising EDTA or a salt thereof.41. The composition of claim 32, further comprising a coating thatinhibits digestion of said composition in a stomach of a subject; anenteric coating; or a gelatin coating.
 42. A method for oraladministration of a recombinant protein having a molecular weight up to100,000 Daltons to a subject, comprising administering orally to saidsubject a pharmaceutical composition comprising said protein; a proteaseinhibitor; and a compound selected from the group consisting of N-(8-[2-hydroxybenzoyl]amino)caprylate (SNAC),N-(10-[2-hydroxybenzoyl]amino)decanoate (SNAD), a salt of said SNAC orsaid SNAD, and a combination thereof.
 43. The method of claim 42,wherein said recombinant protein is an enzyme.
 44. The method of claim42, wherein said recombinant protein is selected from the groupconsisting of insulin, a glucagon, an interferon gamma, an interferonalpha, a growth hormone, an erythropoietin, a GLP-1, a GLP-1 analogue,and granulocyte colony stimulating factor (G-CSF).
 45. The method ofclaim 42, wherein said pharmaceutical composition further comprises anomega-3 fatty acid.
 46. The method of claim 42, wherein said proteaseinhibitor is selected from the group consisting of a serpin, a suicideinhibitor, a transition state inhibitor, a protein protease inhibitor, achelating agent, a Cysteine protease inhibitor, a Threonine proteaseinhibitor, an Aspartic protease inhibitor, and a Metalloproteaseinhibitor.
 47. The method of claim 46, wherein said protease inhibitoris a serpin, and said serpin is a trypsin inhibitor.
 48. The method ofclaim 42, wherein said protease inhibitor is selected from the groupconsisting of Lima bean trypsin inhibitor, Aprotinin, soy bean trypsininhibitor (SBTI), and Ovomucoid.
 49. The method of claim 42, whereinsaid pharmaceutical composition further comprises EDTA or a saltthereof.
 50. The method of claim 42, wherein said pharmaceuticalcomposition further comprises a coating that inhibits digestion of saidpharmaceutical composition in a stomach of a subject; an entericcoating; or a gelatin coating.
 51. A method for treating diabetesmellitus in a subject, comprising administering orally to said subject apharmaceutical composition comprising insulin, Exenatide, or acombination thereof; a protease inhibitor; and a compound selected fromthe group consisting of N-(8-[2-hydroxybenzoyl]amino)caprylate (SNAC),N-(10-[2-hydroxybenzoyl]amino)decanoate (SNAD), a salt of said SNAC orsaid SNAD, and a combination thereof.
 52. The method of claim 51,wherein said insulin or Exenatide is recombinant insulin or Exenatide.53. The method of claim 51, wherein said pharmaceutical compositionfurther comprises an omega-3 fatty acid.
 54. The method of claim 51,wherein said protease inhibitor is selected from the group consisting ofa serpin, a suicide inhibitor, a transition state inhibitor, a proteinprotease inhibitor, a chelating agent, a Cysteine protease inhibitor, aThreonine protease inhibitor, an Aspartic protease inhibitor, and aMetalloprotease inhibitor.
 55. The method of claim 51, wherein saidprotease inhibitor is selected from the group consisting of Lima beantrypsin inhibitor, Aprotinin, soy bean trypsin inhibitor (SBTI), andOvomucoid.
 56. The method of claim 51, wherein said pharmaceuticalcomposition further comprises EDTA or a salt thereof.
 57. The method ofclaim 51, wherein said pharmaceutical composition further comprises acoating that inhibits digestion of said pharmaceutical composition in astomach of a subject; an enteric coating; or a gelatin coating.